Clinical Performance of Immunonephelometric Assay and Chemiluminescent Immunoassay for Detection of IgG Subclasses in Chinese

Abstract

Background
Detection of IgG subclasses (IgGSc) is vital for the diagnosis and management of disease, especially IgG4‐related diseases (IgG4‐RD). This study aimed to evaluate the performances of the chemiluminescent immunoassay (CLIA) for detecting IgGSc and diagnosing IgG4‐RD by IgGSc.

Methods
A total of 40 individuals with IgG4‐RD, 40 with primary Sjogren's syndrome (pSS), and 40 healthy controls (HCs) were enrolled. Serum samples were collected for the simultaneous detection of IgG1, IgG2, IgG3, and IgG4 by the Siemens immunonephelometric assay and the CLIA. The correlation analysis was performed, and diagnostic value was analyzed by the receiver operating characteristic (ROC) curve.

Results
Patients with IgG4‐RD had higher IgG4 (p < 0.001) and lower IgG1 (p < 0.001) than those with pSS, and HC. The results by the Siemens immunonephelometric assay and the CLIA showed a strong correlation in detecting IgG1, IgG2, IgG3, and IgG4 (r = 0.937, r = 0.847, r = 0.871, r = 0.990, all p < 0.001, respectively). The sum of IgG1, IgG2, IgG3, and IgG4 using two assays strongly correlated with total IgG by the IMMAGE 800 (r = 0.866, r = 0.811, both p < 0.001, respectively). For discriminating IgG4‐RD from pSS and HC, no significant differences were observed in CLIA IgG4 and Siemens immunonephelometric assay IgG4 (z = 0.138, p = 0.891), which provided the area under the curves (AUCs) of 0.951 (p < 0.001) and 0.950 (p < 0.001), respectively. The AUCs of CLIA IgG1 and Siemens immunonephelometric assay IgG1 in distinguishing pSS from IgG4‐RD and HC were 0.761 (p < 0.001) and 0.765 (p < 0.001), respectively, with no significant differences (z = 0.228, p = 0.820).

Conclusions
The CLIA and the Siemens immunonephelometric assay appeared to have good consistency with comparable diagnostic value in detecting IgGSc, especially IgG4, and IgG1 that can accurately identify IgG4‐RD or pSS in clinical practice.

Full text

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Journal of Clinical Laboratory Analysis, 2024; 38:e25033
https://doi.org/10.1002/jcla.25033
Journal of Clinical Laboratory Analysis
RESEARCH ARTICLE OPEN ACCESS
Clinical Performance of Immunonephelometric Assay
and Chemiluminescent Immunoassay for Detection of IgG
Subclasses in Chinese
YanQin1,2 | YuhanJia3 | CongcongLiang1 | RuiFu1 | ZhaojunLiang1 | YanlinWang1 | MinFeng1 | ChongGao4 |
JingLuo1
1Department of Rheumatology, the Second Hospital of Shanxi Medical University, Taiyuan, Shanxi, China | 2Shanxi Center for Clinical Laborator y,
Taiyuan, Shanx i, China | 3The Shanxi Medical University, Taiyuan, Shan xi, China | 4Department of Pathology, Brigham and Women's Hospital, Harvard
Medical School, Boston, Massachusetts, USA
Correspondence: Jing Luo (ljty 966@hotmail.com)
Received: 7 June 2023 | Revised: 4 February 2024 | Accepted: 9 March 2024
Funding: This research was supported by the Nature F und Projects of Shanxi Science and Technology Department (201901D111377), the Scientific
Research Project of Health commission of Shanxi Province (2019044), the Research Project Supported by Shanxi Scholarship Council of China (2020-
191), the Science and Technology Innovation Project of Shan xi Prov ince (2020 SYS08), and the Central Government Guides L ocal Science and Technology
Development Fund (YDZJSX2022C031).
Keywords: chemiluminescent immunoassay | IgG subclasses | IgG4- related diseases | immunonephelometric assay | primary Sjogren's syndrome
ABSTRACT
Background: Detection of IgG subclasses (IgGSc) is vital for the diagnosis and management of disease, especially IgG4- related
diseases (IgG4- RD). This study aimed to evaluate the performances of the chemiluminescent immunoassay (CLIA) for detecting
IgGSc and diagnosing IgG4- RD by IgGSc.
Methods: A total of 40 individuals with IgG4- RD, 40 with primary Sjogren's syndrome (pSS), and 40 healthy controls (HCs)
were enrolled. Serum samples were collected for the simultaneous detection of IgG1, IgG2, IgG3, and IgG4 by the Siemens immu-
nonephelometric assay and the CLIA. The correlation analysis was performed, and diagnostic value was analyzed by the receiver
operating characteristic (ROC) curve.
Results: Patients with IgG4- RD had higher IgG4 (p < 0.001) and lower IgG1 (p < 0.001) than those with pSS, and HC. The
results by the Siemens immunonephelometric assay and the CLIA showed a strong correlation in detecting IgG1, IgG2,
IgG3, and IgG4 (r = 0.937, r = 0 .847, r = 0.871, r = 0.990 , all p < 0.001, respectively). The sum of IgG1, IgG2, IgG3, and IgG4
using two assays strongly correlated with total IgG by the IMMAGE 800 (r = 0.866, r = 0.811, both p < 0.0 01, respectively).
For discriminating IgG4- RD from pSS and HC, no significant differences were observed in CLIA IgG4 and Siemens immu-
nonephelometric assay IgG4 (z = 0.138, p = 0.891), which provided the area under the curves (AUCs) of 0.951 (p < 0.001) and
0.950 (p < 0.001), respectively. The AUCs of CLIA IgG1 and Siemens immunonephelometric assay IgG1 in distinguishing pSS
from IgG4- RD and HC were 0.761 (p < 0.001) and 0.765 (p < 0.001), respectively, with no significant differences (z = 0.2 28,
p = 0. 82 0).
Conclusions: The CLIA and the Siemens immunonephelometric assay appeared to have good consistency with comparable
diagnostic value in detecting IgGSc, especially IgG4, and IgG1 that can accurately identify IgG4- RD or pSS in clinical practice.
This is a n open access ar ticle under the terms of t he Creative Commons Attr ibution-NonCommercial-NoDer ivs License, whi ch permits use and d istribution in any me dium, provided th e original
work is properl y cited, the use is non- commercial and no mo difications or a daptations are ma de.
© 2024 T he Authors. Journ al of Clinical Lab oratory Ana lysis published b y Wiley Periodica ls LLC.

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1 | Introduction
Immunoglobulin G (IgG) is the main molecule of human
immunity, accounting for around 75% of the total immuno-
globulin in the total serum of a healthy individual. In a study
analyzing IgG myeloma samples, Terry et al. proposed that
IgG can be divided into different subclasses [1]. At the same
period, another study described human IgG can be divided
into four subclasses: IgG1, IgG2, IgG3, and IgG4, based on the
immunogenicity of distinct heavy chain subgroups [2]. The
physiological characteristics and functions of the four sub-
classes are different, and their functions in immune response
are also variant. In recent years, numerous studies have found
that abnormal levels of IgG subclasses (IgGSc) are associated
with autoimmune diseases, infections, and tumors, especially
in the occurrence and development of IgG4- related diseases
(IgG4- RD), suggesting that dynamic detection of IgGSc has
important clinical application value for the diagnosis, patho-
genesis, and prognosis of these diseases [3, 4].
IgG4- RD is an autoimmune disease with chronic and progres-
sive inflammation and fibrosis, characterized by multiple-
organ involvement, predominating the salivary or lacrimal
glands, retroperitoneal space, lymph node, pancreas, biliary
tract, and thyroid [5]. The diagnosis of IgG4- RD requires a
combination of clinical, serological, radiological, and patho-
logical data. Elevated IgG4 level is the major serological fea-
ture, and serum IgG4 >1350 mg/L is considered as a diagnosis
criterion for IgG4- RD [6]. The IgG4- RD response index (RI)
was a disease activity tool modeled by Stone etal., which also
included serum IgG4 concentration [7]. Some studies show
that serum IgG4 concentrations can provide important clues
for diagnosis and some guidance in the longitudinal assess-
ment of disease activity[8, 9].
At present, analytical methods for IgGSc mainly include the
binding site (TBS) IgGSc assay and immunoturbidimetric assay,
and the Siemens is becoming the most commonly used estab-
lished assay [10,11]. In recent years, the chemiluminescent im-
munoassay (CLIA) has been widely used in clinical diagnosis
and research due to its advantages of sensitivity, rapidity, and
wide linear range [12]. In this study, we report the clinical per-
formance of the CLIA for the measurement of serum IgGSc
based on a comparison with the immunoturbidimetric assay
and have evaluated its application.
2 | Materials and Methods
2.1 | Patients
A total of 40 patients with IgG4- RD (23 females, 17 males, mean
age 59.18 ± 9.30 years) who met the 2019 American College of
Rheumatology/European League Against Rheumatism clas-
sification criteria and 40 patients with primary Sjogren's syn-
drome (pSS) (29 females, 11 males, mean age 57.83 ± 10.18)
who met 2016 American College of Rheumatology/European
League Against Rheumatism classification criteria were en-
rolled from the Second Hospital of Shanxi Medical University
between January 2020 and November 2021 [13, 14]. Peripheral
venous blood samples from patients were collected immedi-
ately after admission. Forty age- and gender- matched healthy
individuals (24 females, 16 males, mean age 57.00 ± 7.37) were
recruited as healthy controls (HCs). This study was approved by
the ethics committee of the Second Hospital of Shanxi Medical
University (2016KY007). Informed consent was obtained from
all individuals.
2.2 | Clinical and Laboratory Indexes
The clinical parameters of all patients were retrospectively col-
lected from clinical records with a predesigned form, including
age, gender, and clinical manifestations of multisystem involve-
ment. In addition, data on the levels of erythrocyte sedimen-
tation rate (ESR), C- reactive protein (CRP), IgG, IgM, and IgA
were also collected. The levels of IgM, IgA, and IgG were mea-
sured using the Beckman Coulter IMMAGE 800.
2.3 | Immunonephelometric IgGSc Assay
The IgG1, IgG2, IgG3, and IgG4 assay kits (immunonephelo-
metric assay; Siemens Healthcare Diagnostics Products GmbH)
and automatic protein analyzer (Siemens BN ProSpec System)
were used for IgGSc assay according to the manufacturer's in-
structions. The protein in the sample forms an immune complex
with the specific antibody, and the resultant immune complex
scatters the light that passes through the specimen, the inten-
sity of which is proportional to the concentration of the relevant
protein in the specimen. The Siemens immunonephelometric
assay provides the qualitative determination of IgG1, IgG2,
IgG3, and IgG4. The reference intervals of IgG1, IgG2, IgG3, and
IgG4 proposed by the manufacturer were 4.05–10.11, 1.69–7.86,
0.11–0.85, and 0.03–2.01 g/L.
2.4 | CLIA IgGSc Assay
CLIA IgGSc assay was performed by the chemiluminescence
immunoanalyzer (YHLO iFlash 3000) and IgG1, IgG2, IgG3,
and IgG4 assay kits (chemiluminescent immunoassay; YHLO
Biotechnology Co., Ltd). IgG1, IgG2, IgG3, and IgG4 were de-
tected by sandwich immunoassay using direct chemilumines-
cence technology. IgG antibody- coated paramagnetic particles
bind to IgG in the sample to form IgG–antibody complex. Under
the action of magnetic field, magnetic particles adsorbed to
the wall of the reaction tube, and the unbonded substance was
washed away by the cleaning solution. Subsequently, the anti-
human IgG antibody acridol marker was added and reacted with
the formed IgG–antibody complex to form the sandwich com-
plex, which was then measured by optical system. The concen-
tration of IgG subclass in the sample was positively correlated
with the relative luminescence intensity. All manufacturer-
proposed reference intervals of IgG1, IgG2, IgG3, and IgG4
were 3941–10,444, 1661–8064, 101–895, and 36–2090 μg/mL,
respectively.
2.5 | Statistical Analysis
All data were analyzed using SPSS 22.0 and GraphPad Prism
7. Data were described as median (Q25, Q75), mean ± SD for
continuous variables. Data from three groups were compared
by one- way analysis of variance (ANOVA) or the independent-
samples Kruskal–Wallis test. The Mann–Whitney U test was

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used to compare the differences between two groups. The
chi- squared test was used to compare the difference in cat-
egorical variables, which were expressed as numbers with
percentages. Spearman's rank correlation was used to calcu-
late the correlation coefficients between the CLIA and the
Siemens immunonephelometric assay. Receiver operating
characteristic (ROC) curves were plotted, and the differences
among the areas under the ROC curves (AUC) were calcu-
lated by the MedCalc 15.2.0 software. p values < 0.05 were
considered to be statistically significant.
3 | Results
3.1 | Demographic and Clinical Characteristics
of IgG4- RD and pSS Patients
The demographic and laboratory features of the study partici-
pants are summarized in Table 1. There were no significant
differences in age (p = 0.555) and gender (p = 0.329) among
IgG4- RD, pSS, and HC. The patients with IgG4- RD exhibited a
lower IgA (p < 0.001) and IgM (p = 0.023) than those with pSS,
but no significant difference in ESR, CRP, TP, ALB, GLO, and
IgG (p = 0.92 8, p = 0. 433, p = 0.551, p = 0.941, p = 0.4 48, p = 0.377,
respectively). The clinical manifestations such as dry mouth,
lung involvement, and thyroid involvement were similar in the
two groups (all p > 0. 05).
3.2 | The Levels of IgG1, IgG2, IgG3, and IgG4 in
IgG4- RD and pSS Patients
As shown in Figure 1, for both Siemens immunonephelomet-
ric assay and CLIA results, the IgG4 levels were significantly
higher in patients with IgG4- RD than those with the pSS, and
HC (p < 0.001). For CLIA results, the levels of IgG1 (10086.50
[8587.75, 12216.75] U/mL vs. 7555.50 [6612.75, 9808.50] U/mL,
p = 0.007), IgG3 (502.00 [146.25, 797.75] U/mL vs. 228.50 [108.00,
360.75] U/mL, p = 0.002), and IgG4 (4567.50 [2564.25, 10087.75]
U/mL vs. 368.50 [174.50, 666.50] U/mL, p < 0.0 01) were signif i-
cantly higher in individuals with IgG4- RD than in HC. This may
suggest that IgG1, IgG3, and IgG4 contribute to the diagnosis of
IgG4- R D. Compared to patients with pSS, patients with IgG4- RD
had significantly reduced IgG1 (10086.50 [8587.75, 12216.75]
U/mL vs. 13810.00 [9927.00, 19343.25] U/mL, p = 0.0038) and
increased IgG4 (4567.50 [2564.25, 10087.75] U/mL vs. 351.00
[125.75, 594.00] U/mL, p < 0.001), which indicated that IgG1 and
IgG4 are helpful in differentiating IgG4- RD from pSS (TableS1).
The Siemens immunonephelometric assay showed the same
trend (TableS2).
TABLE  | Detailed demographic and laborator y features of the enrolled individuals.
IgG4- RD (n = 4 0) pSS (n = 4 0) HC (n = 40) p
Age, years 59.18 ± 9. 30 57.83 ± 10.18 57. 0 0 ± 7. 37 0.555
Female/male 23/17 29/11 2 4/16 0.329
Asians 40 40 40 1.000
Serology, median (IQR)
ES R (m m/h) 27.00 (13.00, 46.00) 23.00 (13.25, 51.50) —0.928
CR P (mg/L) 3.14 (3.13, 6.20) 3.14 (3.13, 13.65) —0.433
TP (g /L) 69.70 (63.40, 74.80) 70.30 (65.60, 75.30) — 0.551
ALB (g /L) 37.70 (34.20, 41.10) 38.20 (33.23, 41.20) —0.941
GLO (g/ L) 32.40 (26.90, 35.50) 31.70 (28.10, 38.20) — 0.448
IgG ( g/L) 15.60 (12.25, 19.00) 14.00 (11.65, 18.88) — 0.377
Ig A ( g/L) 1.9 0 (1.3 0, 2.4 4) 2.91 (2.05, 4.14) —< 0.0 01
IgM (g/L) 0.92 (0.55, 1.23) 1.05 (0.73, 1.62) —0.023
Organ involvement, n (%)
Dry mouth 27 (67.50) 33 (82.50) —0.196
Dry eye 17 (42.50) 27 (67.50) —0.042
Pancreas 6 (15.00) 0 — NA
Bile duct 2 (5.0 0) 0 — NA
Lung 11 (27.5 0) 5 (12.50) —0.099
Kidney 6 (15.0 0) 0 — NA
Retroperitoneum 4 (10.00) 0 — NA
Paranasal sinus 4 (10.0 0) 0 — NA
Lymph node 10 (25.00) 1 (2.50) —0.007
Thyroid 5 (15.0 0) 4 (10.00) —0.348
Note: All data were descr ibed as median (interquartile range) or numbers (percentage) except age, which were compared using the Kruskal–Wallis one- way ANOVA
test or the Mann–Whitney U test. Dif ference in categorical va riables was compared by the chi- squa red test. Age was compared using the independent sample t- test.
Bold values indicate statistically significant (p < 0.05).
Abbreviations: ALB, albumin; CLIA , chemiluminescent immunoassay ; CRP, C- reactive protein; ESR, ery throcyte sedimentation rate; GLO, globuli n; HC, healthy
control; IgG, immunoglobulin; IgG 4- RD, IgG4- related disea ses; NA, not available; pSS, primary Sjogren's syndrome; TP, total protein.

4 of 12 Journal of Clinical Laboratory Analysis, 2024
3.3 | Correlations Between the Levels of IgGSc
Determined by the YHLO CLIA and Siemens
Immunonephelometric Assays
We next analyzed the correlation between the Siemens immu-
nonephelometric assay and the YHLO CLIA (Figure 2). The
correlation coefficients of IgG1, IgG2, IgG3, and IgG4 detected
by the CLIA and the Siemens immunonephelometric assay
were 0.937, 0.847, 0.871, and 0.990 (all p < 0.001), respectively,
which indicated that the levels of IgGSc determined by the
two assays exhibited good correlations. The correlation coef-
ficient of sum of IgG1, IgG2, IgG3, and IgG4 detected by the
CLIA and the Siemens immunonephelometric assay was 0.945
(p < 0 .0 01) .
We also evaluated the proportion of patients with IgG4- RD,
pSS, and HC whose serum IgG1, IgG2, IgG3, and IgG4 levels ex-
ceeded the upper limit determined by the two assays. As shown
in Figure 3 and TableS3, there were no significant differences in
the detection rates of IgG1, IgG2, IgG3, and IgG4 in patients with
IG4- RD and pSS, and in HC between the two assays (all p > 0.05) .
3.4 | Relationships Between the Sum of IgGSc
and the Total Serum IgG Measured Using
the Beckman Coulter IMMAGE 800
We further investigated the correlation between the sum of
IgGSc and the total serum IgG measured using the Beckman
FIGUR E  | Serum levels of IgG subclass in patients with IgG4- RD and pSS measured by the Siemens assay and CLIA. IgG subclasses measured
using the (a) CLIA (b) and the Siemens BN P. Data were presented as median (Q25, Q75) and were analyzed by the Kruskal–Wallis test. *p < 0 .05,
**p < 0.01, ***p < 0. 001.

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Coulter IMMAGE 800 in patients with IgG4- RD and pSS
(Figure4). The strong positive correlations were found between
the sum of IgG1, IgG2, IgG3, and IgG4 detected by the CLIA and
the Siemens immunonephelometric assay and the total serum
IgG (r = 0.811, p < 0.001; r = 0.866, p < 0.001, respectively). The
good correlation between the sum of the IgG1, IgG2, IgG3, and
IgG4 and the measured value of total IgG supported the accu-
racy of the two assays.
3.5 | Cutoff Values for the YHLO CLIA
and the Siemens Immunonephelometric Assay
Although a good correlation between the results w ith two methods
were found, the measured v alues were different. Therefore, the cut-
off values also varied depending on the reagent used. We next cal-
culated the IgG4- RD cutoff values for the two IgG4 methods. ROC
analysis was performed between patients with IgG4- RD, pSS and
FIGUR E  | Correlation of serum IgG subclasses levels detected by the Siemens BN P to those by the CLIA. Correlation of serum IgG1 (a), IgG2
(b), IgG3 (c), IgG4 (d), and the sum of IgG1, IgG2, IgG3, and IgG4 (e) detected by the Siemens BN P to those by the CLIA. Data were analyzed by
Spearman's rank correlation.

6 of 12 Journal of Clinical Laboratory Analysis, 2024
in HC. The cutoff value and AUC, sensitivity, and specificity for
each assay are shown in Tables2 and 3. The cutoff values of CLIA
IgG4 and Siemens immunonephelometric assay IgG4 in discrimi-
nating IgG4- RD from pSS were 1804 μg/mL and 1490, respectively,
and the cutoff values for distinguishing IgG4- RD from HC were
1126 μg/mL and 1100, respectively. The cutoff values of CLIA IgG1
and Siemens immunonephelometric assay IgG1 in discriminat-
ing pSS from HC were 11,269 and 7880 μg/mL, respectively. The
cutoff value of CLIA IgG3 in discriminating pSS from HC was
408 μg/mL, which was close to the cutoff value of 472 μg/mL of the
Siemens immunonephelometric assay.
3.6 | Clinical Values of IgGSc Determined
by the Siemens Immunonephelometric Assay
and the YHLO CLIA in Diagnosing IgG4- RD
and pSS
Based on Figure 1, the diagnostic performance of IgGSc deter-
mined by the CLIA and the Siemens immunonephelometric assay
was further evaluated, and the results are summarized in Tables2
and 3. Although the cutoff values differed between the two meth-
ods, no significant differences in AUC, sensitivity, and specificity
were observed . We dr ew ROC curves to compare the di fference be-
tween two assays in distinguishing specific patients and controls
(Figures5 and 6). The AUCs of CLIA IgG4 and Siemens immu-
nonephelometric assay IgG4 were 0.951 (95% confidence interval
[CI]: 0.895–0.982) and 0.950 (95% CI: 0.894–0.981) in discriminat-
ing IgG4- RD from pSS and HC, and the AUC for distinguishing
IgG4- RD from pSS were 0.953 (95% CI: 0.881–0.988) and 0.952
(95% CI: 0.879–0.987), and the AUCs for distinguishing IgG4- RD
from HC were 0.949 (95% CI: 0.875–0.986) and 0.948 (95% CI:
0.874–0.985), with no significant difference (z = 0.138, p = 0.8 91;
z = 0.144, p = 0.885; z = 0.138, p = 0.891; z = 0.103, p = 0 .918, respe c-
tively). In addition, CLIA IgG1, Siemens immunonephelometric
assay IgG1, CLIA IgG3, and Siemens immunonephelometric
assay IgG3 can be used to distinguish IgG4- RD from HC, with
AUCs of 0.721 (95% CI: 0.609–0.815), 0.812 (95% CI: 0.709–0.891),
0.712 (95% CI: 0.600–0.808), and 0.735 (95% CI: 0.624–0.827),
FIGUR E  | Comparison of the positive rate of IgG subclasses measured by the Siemens BN P and the CLIA in different cohorts. The positive rate
of IgG subclasses in patients with (a) I gG4- RD and (b) pSS. The positive rate of IgG subclas ses (c) in the HC group and (d) in all i ndividuals. Data were
analyzed by the chi- squared test. NS: p > 0.05 .

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respectively. For IgG1, a significantly higher AUC was found with
the Siemens immunonephelometric assay than with the CLIA
(z = 2.791, p = 0.005), and there was no significant difference in
the AUCs between CLIA IgG3 and Siemens immunonephelomet-
ric assay IgG3 (z = 0.561, p = 0.5755) (Figure5).
The AUCs of CLIA IgG1 and Siemens immunonephelometric
assay IgG1 were 0.761 (95% CI: 0.675–0.834) and 0.765 (95% CI:
0.679–0.838) for discriminating pSS from IgG4- RD and HC,
and the AUCs of CLIA IgG4 and Siemens immunonephelomet-
ric assay IgG4 were 0.745 (95% CI: 0.658–0.821) and 0.746 (95%
CI: 0.658– 0.821), without a significant difference (z = 0.228,
p = 0.820; z = 0.056, p = 0.956, respectively). In discriminating
pSS from and HC, there were no significant differences in the
AUCs between CLI A IgG1 and Siemens immunonephelometric
assay IgG1 or CLIA IgG3 and Siemens immunonephelometric
assay IgG3 (z = 1.625, p = 0.104; z = 0.465, p = 0.642, respec-
tively). In conclusion, these results indicate that the CLIA has a
similar diagnostic value to the Siemens immunonephelometric
assay (Figure6).
4 | Discussion
Polyclonal hypergammaglobulinemia (PHGG) is a physiologi-
cal reaction to liver disease, hematological disorders, infection,
inflammation, and so on and is characterized by the overpro-
duction of immunoglobulins by plasma cells and high levels of
IgGSc [15, 16]. Autoimmune diseases such as pSS, rheumatoid
arthritis, and eosinophilic granulomatosis with polyangiitis can
also cause PHGG [17]. In addition to lymphadenopathy and eo-
sinophilia, PHGG also is the hematological manifestations of
FIGUR E  | Relationships between the sum of IgG1, IgG2, IgG3, and IgG4 and the total serum IgG measured by the Beckman Coulter IMMAGE
800 in patients with IgG4- R D and pSS. Relationship between the serum total IgG and the sum of IgG subclasses measured using the (a) CLIA and
(b) Siemens BN P.

8 of 12 Journal of Clinical Laboratory Analysis, 2024
TABLE  | Diagnostic accuracy of IgG subclass to distinguish patients with IgG4- RD from those with pSS and HCs.
AUC (95% CI) p
Cutoff
(µ g/mL) Sen (95% CI) Spe (95% CI) +LR (95% CI) LR (95% CI) z p′
IgG4- RD versus pSS+HC
CLIA- IgG4 0.951 (0.895, 0.982) < 0.0 01 > 180 4 87.50 (73.20, 95.80) 96.25 (89.40, 99.20) 23.33 (7.60,71.3) 0.13 (0.06, 0.30) 0.138 0.891
Siemens- IgG4 0.950 (0.894, 0.981) < 0.0 01 > 149 0 87.50 (73.20, 95.80) 95.00 (87.70, 98.60) 17.50 (6.70, 45.80) 0.13 (0.06, 0.30)
IgG4- RD versus pSS
CLIA- IgG4 0.953 (0.881, 0.988) < 0.0 01 > 180 4 87.50 (73.20, 95.80) 97.50 (86.80, 99.90) 35.00 (5.00, 243.3) 0.13 (0.06, 0.30) 0.144 0.885
Siemens- IgG4 0.952 (0.879, 987) < 0 .001 > 1490 87.50 (73.20, 95.80) 95.00 (83.10, 99.40) 17.50 (4 .50 , 67.9 0) 0.13 (0.06, 0.30)
IgG4- RD versus HC
CLIA- IgG1 0.721 (0.609, 0.815) < 0 .001 > 7656 80.00 (64.40, 90.90) 55.00 (38.50, 70.70) 1.78 (1.20, 2.60) 0.36 (0.20, 0.70) 2.791 0.005
Siemens- IgG1 0.812 (0.709, 0.891) < 0.0 01 > 8120 77.50 (61.50, 89.20) 80.00 (64.40, 90.90) 3.88 (2.00, 7.40) 0.28 (0.20,0.50)
CLIA- IgG3 0.712 (0.600, 0.808) < 0.0 01 > 408 65.00 (48.30, 79.40) 85.00 (70.20, 94.30) 4.33 (2.00, 9.40) 0.41 (0.30, 0.60) 0.561 0.575
Siemens- IgG3 0.735 (0.624, 0.827) < 0.0 01 > 361. 6 67.50 (50.90, 81.40) 70.00 (53.50, 83.40) 2.25 (1.30, 3.80) 0.46 (0.30, 0.80)
CLIA- IgG4 0.949 (0.875, 0.986) < 0.0 01 > 1126 87.50 (73.20, 95.80) 95.00 (83.10, 99.40) 17.50 (4.5 0, 67.90) 0.13 (0.06, 0.30) 0.103 0.918
Siemens- IgG4 0.948 (0.874, 0.985) < 0.0 01 > 110 0 87.50 (73.20, 95.80) 95.00 (83.10, 99.40) 17. 50 (4 .50 , 67.9 0) 0.13 (0.06, 0.30)
Abbreviations: AUC, area under roc cur ve; CI, confidence interval; L R, likelihood ratio; Sen, sensitivity; Spe, specif icity.
TABLE  | Diagnostic accuracy of IgG subclass to distinguish patients with pSS from those with IgG4- RD and HCs.
AUC (95% CI) p
Cutoff
(µ g/mL) Sen (95% CI) Spe (95% CI) +LR (95% CI) LR (95% CI) z p′
pSS versus IgG4- RD+HC
CLIA- IgG1 0.761 (0.675, 0.834) < 0. 001 > 12,303 62.50 (45.80, 77.30) 90.00 (81.20, 95.60) 6.25 (3.10, 12.60) 0.42 (0.30, 0.60) 0.228 0.820
Siemens- IgG1 0.765 (0.679, 0.838) < 0.0 01 > 10, 28 0 62.50 (45.80, 77.30) 83.75 (73.80, 91.10) 3.85 (2.20, 6.70) 0.45 (0.30, 0.70)
CLIA- IgG4 0.745 (0.658, 0.821) < 0 .001 ≤ 18 04 97.50 (86.80, 99.90) 46.25 (35.00, 57.80) 1.81 (1.50, 2.20) 0.05 (0.01, 0.40) 0.056 0.956
Siemens- IgG4 0.746 (0.658, 0.821) < 0.0 01 ≤ 1820 97.50 (86.80, 99.90) 45.00 (33.80, 56.50) 1.77 (1.40, 2.20) 0.06 (0.01, 0.40)
pSS versus HC
CLIA- IgG1 0.838 (0.738, 0.911) < 0. 001 > 11,2 69 70.00 (53.50, 83.40) 92.50 (79.60, 98.40) 9.33 (3.10, 28.20) 0.32 (0.20, 0.50) 1.625 0.104
Siemens- IgG1 0.870 (0.776, 0.935) < 0.0 01 > 7880 85.00 (70.20, 94.30) 77.50 (61.50, 89.20) 3.78 (2.10, 6.80) 0.19 (0.09, 0.40)
CLIA- IgG3 0.723 (0.611, 0.817) < 0. 001 > 40 8 55.00 (38.50, 70.70) 85.00 (70.20, 94.30) 3.67 (1.70, 8.10) 0.53 (0.40, 0.80) 0.465 0.642
Siemens- IgG3 0.738 (0.628, 0.830) < 0 .001 > 472 55.00 (38.50, 70.70) 90.00 (76.30, 97.20) 5.50 (2.10, 14.50) 0.50 (0.30, 0.70)

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IgG4- RD [18]. Moreover, PDHH can provide important clues for
IgG4- RD with variable clinical manifestations and difficult di-
agnosis; in particular, serum IgG4 concentration can be used as
a screening index for IgG4- RD [19]. Serum protein electropho-
resis, quantitative immune nephelometry, and IgGSc assay are
important for immunoglobulin detection of PDHH. Sometimes,
serum protein electrophoresis is performed in conjunction with
IgGSc detection to determine whether elevated immunoglobu-
lins are polyclonal [20].
In addition, the measurement of IgGSc is widely performed as
part of the laboratory evaluation of immunologic deficiencies,
autoimmune diseases, infections, and tumors [21]. Especially
for IgG4- RD, circulating IgGSc is strongly associated with their
FIGUR E  | Receiver operating characteristic curve (ROC) analysis of the serum levels of IgG subclasses. (a) ROC of IgG4 levels to distinguish
patients with IgG4- R D from those with (a) pSS and HCs, (b) pSS. (c) ROCs of IgG1, IgG3, and IgG4 to distinguish patients with IgG4- RD patients
from those with HCs.

10 of 12 Journal of Clinical Laboratory Analysis, 2024
occurrence and development, prognosis, or treatment [22]. In
clinical laboratories, quantitative detection of IgGSc profile
(IgG1, IgG2, IgG3, and IgG4) is generally performed by the
Siemens immunonephelometric assay [23]. In recent years, liq-
uid chromatography- mass spectrometry (LC–MS) has rapidly
gained attention as a method for the identification and monitor-
ing of serum immunoglobulin and has also become a method
for the detection of IgGSc. Van der Gugten etal., for the perfor-
mance of LC–MS for the detection of IgGSc, demonstrated that
LC–MS could compensate for the inconsistency of the Siemens
immunonephelometric assay in the detection of IgG2 [24]. In
addition, the CLIA also is a convenient and commonly used
method for quantitative analysis of protein targets in laborato-
ries. However, comprehensive validation and performance of the
CLIA for IgGSc has not been done. Since reliable experimental
performance is important for the clinical application of diagnos-
tic markers, it is necessary to perform reliable performance ver-
ification for the newly developed commercial analytical assays.
In this study, we used the automated CLIA to measure IgGSc val-
ues in patients with IgG4- RD and pSS versus healthy adults, com-
pared with the Siemens immunonephelometric assay. Our results
overall showed a good correlation to the CLIA and the Siemens
immunonephelometric assay. We analyzed the diagnostic value
of the CLIA for the detection of IgGSc and calculated the cutoff
value for each reagent. The AUCs between the two methods were
compared between patients with IgG4- RD and those with pSS,
which need to be distinguished from IgG4- RD. The applicability
of the CLIA for IgGSc was investigated using serum of patients
with IgG4- RD, patients with pSS, and HC. The results revealed
significantly higher levels of IgG1, IgG3, and IgG4 among patients
with IgG4- RD and pSS than those of in HC. In daily clinical prac-
tice, serum IgG4 level is considered an important marker for the
diagnosis of IgG4- RD. It is worth noting that IgG and IgG subclass
levels vary by race. Some studies have demonstrated that the con-
centrations of IgG and IgG4 are higher in Asian people than in
White people with the IgG4- RD, which has the potential to affect
the interpretation of these diagnostic tests in the clinical setting
and in research [25, 26]. In this study, all the individuals included
were Chinese, and there was no racial difference.
In addition, there were good correlation in IgG1, IgG2, IgG3,
and IgG4 between the results with two methods (r = 0 .93 7,
r = 0.847, r = 0.871, r = 0.990, respectively); which manifests
that the quantitative levels of IgGSc measured using the CLIA
were consistent with the levels measured using the Siemens
immunonephelometric assay. The good correlation between
the sum of IgG1, IgG2, IgG3, and IgG4 using the Siemens
FIGUR E  | ROC analy sis of the serum levels of IgG subcla sses. (a) ROC s of IgG1 and IgG4 to disting uish patients with pSS from those with Ig G4-
RD and HCs. (b) ROCs of IgG1 and IgG3 to distinguish patients with pSS from those with HC.

11 of 12
immunonephelometric assay and the CLIA and the total IgG
by IMM AGE 800 were found (r = 0.866, r = 0.811, respect ively),
which supports the accuracy of the determinations. However,
the measured values were different. This disparity may be ex-
plained by some reasons. The first reason may be the varia-
tion in IgGSc levels is associated with different reagents used
for detection. The measurements have not been unified, and
each reference material of each IgGSc values was determined
by different manufacturer, which means that two assays have
established the different traceability. Importantly, IgG1, IgG2,
IgG3, and IgG4 are not monoclonal, and serum IgGSc in pa-
tients is polyclonal with reactivity to multiple antigens [27].
Also, there is currently no consensus on calibration materials
that can be used to detect IgGSc [28]. Another reason is that
we recruited 120 individuals, the small number of sample size.
These factors may have led to the difference between our re-
sults and other reports. Differences in IgGSc values between
the Siemens immunonephelometric assay and other methods,
such as TBS, N- assay LA Nittobo (Nittobo), and BS- NIA have
been previously reported [29].
In clinical practice, it is necessary for each laboratory to develop
its own reference limits. Our study clearly confirms that the
cutoff values of serum IgGSc are not constant for the diagno-
sis of IgG4- RD, and the cutoff value level is dependent on the
population to be identified. As pSS were found to be associated
with elevated IgG4 concentrations [30], we determined the op-
timal cutoff values of CLIA IgG4 (1804 μg/mL) and Siemens
IgG4 (1490 μg/mL) by ROC curve for Chinese population in
differentiating the IgG4- RD group from pSS and HCs, which
will improve our understanding of the laboratory indicators and
improve clinical diagnosis. Under the set cutoff value, the sen-
sitivity of CLI A IgG4 and Siemens immunonephelometric assay
IgG4 were 87.50% and 87.50%, respectively, and specificity were
96.25% and 95.00%, respectively. While the stark difference in
cutoff levels of the diagnostic serum IgGSc between the two as-
says is evident, no significant differences in AUC, sensitivity,
and specificity were observed.
To the best of our knowledge, this study is the first to evaluate the
Siemens immunonephelometric assay versus the YHLO CLIA for
quantitative determination of IgGSc profile (IgG1, IgG2, IgG3, and
IgG4). We showed that the CLIA is a quantitative assay with a
working performance comparable to the Siemens immunoneph-
elometric assay. Quantitative results from the CLIA correlated
good with the Siemens immunonephelometric assay. In addi-
tion, the sum of IgGSc by the CLIA is comparable to that by the
Siemens immunonephelometric assay. Although the good correla-
tion coefficient for all data was also observed between the CLIA
and the Siemens immunonephelometric assay, it does not mean
the CLIA can replace the Siemens immunonephelometric assay.
Clinicians should further evaluate and choose appropriate meth-
ods and proper cutoff values to avoid misclassification of patients.
Furthermore, clinicians should be circumspect in detecting IgGSc
levels using dif ferent reagents to avoid miscalculation of the results
and misclassification of patients. This study can provide a basis
for the CLIA to detect IgGSc to a certain extent and supplement
more choices for the clinical detection of IgGSc. However, multi-
center prospective clinical studies with large sample sizes are nec-
essary to more clearly establish cutoff values of serum IgGSc for
the diagnosis of patients with IgG4- RD or pSS, which is where the
shortcomings of our current study lie and where future research
should be directed.
5 | Conclusion
In summar y, we conclude that IgGSc assays by the CLIA is fea-
sible, which will facilitate the diagnosis of IgG4- RD and pSS,
and be of high value in applications for monitoring disease.
Author Contributions
Yan Qin analyzed the data, and developed and wrote the manuscript.
Yuhan Jia participated in the sample collection and data extraction.
Congcong Liang and Rui Fu performed the experimental procedures of
IgG subclasses. Zhaojun Liang and Yanlin Wang contributed to the analy-
sis and interpret ation of data. Min Feng participated in the statistical anal-
ysis. Chong Ga o provided signific ant revisions to the manusc ript. Jing Luo
generated themes, and guided and supervised throughout the study. All
authors were involved in drafting the article or revising it critically for im-
portant int ellectual content and approved the f inal version to be published .
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
All data generated or analyzed during this study are available from the
corresponding author.
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Supporting Information
Additional supporting information can be found online in the
Supporting Information section.